Continuous fractionated irradiation with irradiation-free intervals enhances survival and clonogenicity in osteosarcoma MG-63 cells via adaptive DNA damage response

Radiotherapy is a widely used treatment modality for various types of cancer. However, the adaptive resistance of tumor cells during radiotherapy poses a major challenge to therapeutic efficacy. This study aimed to evaluate whether continuous fractionated irradiation induces radioresistance in osteosarcoma MG-63 cells compared with single-dose exposure. To assess the effects of fractionated irradiation on cell survival, MG-63 cells were subjected to either single irradiation (SR; 0, 5, or 10 Gy) or continuous fractionated irradiation (5-CFR; 0, 1, or 2 Gy per day for five consecutive days), resulting in total doses of 0, 5, or 10 Gy, respectively. Compared with SR, 5-CFR significantly increased survival and promoted the formation of larger colonies, indicating enhanced clonogenicity. We further examined the effects of additional irradiation (AR) following 5-CFR and an irradiation-free interval. Cells pretreated with 5-CFR (0, 1, or 2 Gy) were subsequently exposed to a single dose of AR (2 Gy), resulting in total doses of 0, 7, or 12 Gy, respectively. MG-63 cells that received 5-CFR + AR exhibited significantly greater survival and increased colony size compared to those treated with SR + AR. To explore the cellular response to DNA damage following 5-CFR, we analyzed γ-H2AX and 53BP1 foci formation. Both markers increased in a dose-dependent manner after 5-CFR, suggesting effective recognition and repair of DNA double-strand breaks. Collectively, these results indicate that continuous fractionated irradiation with irradiation-free intervals confers greater radioresistance to MG-63 cells by enhancing survival and clonogenicity via an adaptive DNA damage response compared with SR.